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Method for detecting bursaphelenchus xylophilus by loop-mediated isothermal amplification reaction

A technology of pine wood nematodes and detection methods, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of poor repeatability, time-consuming, radioactivity, etc.

Pending Publication Date: 2017-08-15
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Abstract
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  • Application Information

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Problems solved by technology

Among them, although DNA probe technology can effectively identify B. xylophilus, it is expensive and radioactive; RAPD technology analyzes the relationship between B. xylophilus and related species, and the result has poor repeatability; RFLP technology distinguishes B. xylophilus from P. Styloid nematodes, endonucleases are expensive and time-consuming; fluorescent PCR is sensitive and fast, but expensive; PCR technology is easy to operate, less expensive, but takes a long time

Method used

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  • Method for detecting bursaphelenchus xylophilus by loop-mediated isothermal amplification reaction
  • Method for detecting bursaphelenchus xylophilus by loop-mediated isothermal amplification reaction
  • Method for detecting bursaphelenchus xylophilus by loop-mediated isothermal amplification reaction

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Embodiment 1

[0038] Example 1 Establishment of a LAMP rapid detection method for Monochamus alternatus carrying pine xylophilus

[0039] 1. Materials and methods

[0040] 1.1 Extraction of Monochamus alternatus DNA

[0041] Take 0.2 g of head and thorax tissues of Monochamus alternatus adults, put them into a 2 mL EP tube, put them into a steel ball mill and grind them for 5 min (60 Hz); add 600 μL of 3M guanidine isothiocyanate extraction buffer, lyse at room temperature for 1 h, and mix by inversion during the period. Mix well; add 600 μL Tris-phenol chloroform isoamyl alcohol (25:24:1), centrifuge at 12000 r / min for 10 min; absorb the supernatant, add 600 μL chloroform isoamyl alcohol (24:1), centrifuge at 12000 r / min for 10 min; absorb the supernatant Add 600 μL of isopropanol to precipitate at -20°C for 20 minutes, centrifuge at 12,000 r / min for 10 minutes; discard the supernatant, wash the precipitate twice with 70% ethanol, and centrifuge at 12,000 r / min for 3 minutes each time; bl...

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Abstract

The invention discloses a method for detecting bursaphelenchus xylophilus by a loop-mediated isothermal amplification reaction, which belongs to the field of molecular detection of bursaphelenchus xylophilus. The SYG-2 gene of bursaphelenchus xylophilus is taken as a target gene, a LAMP primer group with high specificity on the bursaphelenchus xylophilus is obtained by designing and screening, and the LAMP primer group can specifically distinguish the bursaphelenchus xylophilus and bursaphelenchus mucronatus. The LAMP primer group is used for establishing a LAMP rapid detection method for bursaphelenchus xylophilus, and the method can rapidly and accurately discriminate whether a sample has the bursaphelenchus xylophilus or not. The invention further discloses a LAMP detection kit for the bursaphelenchus xylophilus. The method has the advantages of high specificity, simple operation, short time consuming, and low cost, and is suitable for rapid detection of bursaphelenchus xylophilus carried by monochamus alternates and suitable for monitoring the generation and propagation of the bursaphelenchus xylophilus disease.

Description

technical field [0001] The invention relates to molecular detection of pine wood nematodes, in particular to a LAMP detection method for pine wood nematodes carried by Monochamus alternatus, and belongs to the field of molecular detection of pine wood nematodes carried by Monochamus alternatus. Background technique [0002] Pine wilt disease, also known as pine wilt disease and fusarium wilt, is caused by the pine wood nematode [Bursaphelenchus xylophilus (Steiner&Buhrer) Nickle], mainly through insects of the genus Monochamus on pine trees. Natural transmission by feeding and spawning, and long-distance transmission by transportation of diseased wood and diseased wood processed wood. The spread of pine xylophilus in the forest area is mainly by Monochamus alternatus, which can damage Pinus spp., Larix spp., Picea spp., Pseudotsuga spp. .), 36 species of pines and 8 species of non-pines in the genera Abies (Abies spp.) and Cedrus (Cedrus spp.), among which pines were the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119
Inventor 王曦茁汪来发孟繁丽刘洪剑林彩丽
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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